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- PDB-9klq: Cryo-EM structure of ChCas12b-sgRNA-extended non-target DNA terna... -

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Basic information

Entry
Database: PDB / ID: 9klq
TitleCryo-EM structure of ChCas12b-sgRNA-extended non-target DNA ternary complex (Complex-D)
Components
  • C2c1 CRISPR-Cas endonuclease RuvC-like domain-containing protein
  • Non-target DNA strand
  • Target DNA strand
  • sgRNA
KeywordsRNA BINDING PROTEIN/RNA/DNA / CRISPR / Cas12b / gene-editing / RNA BINDING PROTEIN-RNA-DNA complex
Function / homology: / : / C2c1 CRISPR-Cas endonuclease, RuvC-like domain / DNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100) / C2c1 CRISPR-Cas endonuclease RuvC-like domain-containing protein
Function and homology information
Biological speciesCandidatus Hydrogenedentes bacterium ADurb.Bin170 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.11 Å
AuthorsLi, Y. / Li, J. / Pei, X. / Gan, J. / Lin, J.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)32130063 China
CitationJournal: Nucleic Acids Res / Year: 2025
Title: Catalytic-state structure of Candidatus Hydrogenedentes Cas12b revealed by cryo-EM studies.
Authors: Ye Li / Jian Li / Xiaotong Pei / Jingjing Wei / Jianhua Gan / Jinzhong Lin /
Abstract: The CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein) systems are adaptive immune mechanisms that play critical roles in defending archaea and ...The CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein) systems are adaptive immune mechanisms that play critical roles in defending archaea and bacteria against invading entities. These systems can be divided into two classes, with class 2 comprising three types (II, V, and VI). Because of their ability to cleave double-stranded DNA, many class 2 CRISPR-Cas proteins have been harnessed as genome editing tools. Unlike the well-studied type II Cas9 proteins, the structural studies of the type V-B Cas12b proteins are limited, hindering their engineering and broader application. Here, we report four complex structures of ChCas12b, which reveal many unique structural features. The folding of the single guide RNA (sgRNA) of ChCas12b is distinct from that of AacCas12b and BthCas12b. Notably, many of these unique features are involved in ChCas12b-sgRNA interaction, suggesting that they are co-evolved. While ChCas12b shares a conserved two-cation-assisted catalytic mechanism with its homologs, it recognizes a longer guide:target heteroduplex, potentially offering higher fidelity in DNA editing. Altogether, our studies suggested that Cas12b family proteins exhibit significant diversity in their folding, sgRNA and target DNA binding. In the future, it is worth characterizing more representative proteins to identify CRISPR-Cas proteins with higher gene editing ability and fidelity.
History
DepositionNov 14, 2024Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Jul 2, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: C2c1 CRISPR-Cas endonuclease RuvC-like domain-containing protein
B: sgRNA
C: Target DNA strand
D: Non-target DNA strand
hetero molecules


Theoretical massNumber of molelcules
Total (without water)235,7746
Polymers235,7264
Non-polymers492
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein C2c1 CRISPR-Cas endonuclease RuvC-like domain-containing protein


Mass: 167419.828 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Candidatus Hydrogenedentes bacterium ADurb.Bin170 (bacteria)
Gene: BWY07_02509 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A1V5YSD0
#2: RNA chain sgRNA


Mass: 37052.035 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Candidatus Hydrogenedentes bacterium ADurb.Bin170 (bacteria)
#3: DNA chain Target DNA strand


Mass: 15569.955 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Candidatus Hydrogenedentes bacterium ADurb.Bin170 (bacteria)
#4: DNA chain Non-target DNA strand


Mass: 15684.010 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Candidatus Hydrogenedentes bacterium ADurb.Bin170 (bacteria)
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
Has ligand of interestN
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: ChCas12b-sgRNA-extended non-target DNA ternary complex (Complex-D)
Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Source (natural)Organism: Candidatus Hydrogenedentes bacterium ADurb.Bin170 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1500 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

CTF correctionType: NONE
3D reconstructionResolution: 3.11 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 47756 / Symmetry type: POINT

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